A novel bacterium was isolated from a freshwater hot spring, the Hale House Spring, located at Hot Springs National Park, Hot Springs, AR, USA. Cells of strain MP-01T stained Gram-negative, were rod-shaped, non-motile, strictly anaerobic and chemo-organotrophic and did not form spores. Growth occurred at 50–65 °C, with an optimum at 60 °C, at pH 6.0–8.0, with an optimum at pH 6.5–7.0, and at NaCl concentrations up to 0.5?% (w/v), with optimum growth in the absence of NaCl. Strain MP-01T was capable of fermentative growth on pyruvate or proteinaceous substrates as well as reducing Fe(III) and Mn(IV). Major fatty acids were iso-C15?:?0, iso-C16?:?0, anteiso-C17?:?0 and iso-C17?:?0. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine and the major isoprenoid quinone was MK-10. In the polyamine pattern, sym-homospermidine was the predominant compound. The DNA G+C content was 62.7 mol%. Analysis of the 16S rRNA gene sequence of the isolate indicated that strain MP-01T represents the first reported cultivated member of subdivision 23 of the Acidobacteria. It is proposed that strain MP-01T represents a novel genus and species, for which the name Thermoanaerobaculum aquaticum gen. nov., sp. nov. is proposed. The type strain of Thermoanaerobaculum aquaticum is MP-01T (?=?DSM 24856T?=?JCM 18256T).